1. Field of the Invention
The present invention relates to a method for malaria testing, and a reagent or kit for testing, which can be used in the test method.
2. Related Art
Malaria is an infectious disease caused by infection with a malaria parasite, which is mediated by mosquitoes belonging to the genus Anopheles. Malaria parasites (Plasmodium spp.) are protozoa belonging to Apicomplexa, the members of which are obligate intracellular parasites, in other words, they can proliferate only in cells. Protozoan parasites exhibit comparatively high host specificity, and there are four kinds of malaria parasites that utilize humans as a definitive host, i.e., falciparum malaria parasites (P. falciparum), tertian malaria parasites (P. vivax), ovale malaria parasites (P. ovale), and quartan malaria parasites (P. malariae). Malaria caused by these four kinds of malaria parasites are referred to as falciparum malaria, tertian malaria, ovale malaria, quartan malaria, respectively.
It is estimated that the number of patients suffering from malaria is three to five hundred million per year throughout the world, with a death toll of 1.5 to 2.7 million per year. In addition, malaria parasites are indigenous to 100 or more countries predominantly in tropical and subtropical areas, and thus half the global population constantly faces the risk of malaria infection. Therefore, early detection and early treatment are important for preventing increases in the seriousness of the symptoms, and also for avoiding spreading of the infection.
As conventional methods for malaria testing (definitive diagnostic methods), which are widely carried out around the world, a Giemsa-stained thin-film smear preparation and a Giemsa-stained thick-film smear preparation of the blood collected from a subject are prepared, and the malaria-parasitized erythrocytes are then detected by microscopic observation of these preparations (see Non-patent Documents 1 and 2).
Furthermore, a test method in which a reagent for detecting malaria-parasitized erythrocytes is used which contains a DNA selective fluorescent dye, and a reagent for partial lysis of erythrocyte membrane capable of allowing the fluorescent dye to permeated into the erythrocyte while keeping the malaria parasites within the erythrocytes has also been proposed (see Patent Document 1). In this method, the blood collected from a subject and the reagent for detecting malaria-parasitized erythrocytes are mixed to prepare a measurement sample, and an excitation light is irradiated onto this measurement sample to identify the erythrocytes parasitized with malaria, based on the obtained scattering ray data and fluorescent data.
For the early detection and early treatment of malaria, or for disseminating the testing itself of malaria, it is necessary that the test method can be carried out in a convenient manner even in endemic areas of malaria. However, in the method described above including microscopic observation of a smear preparation, each of the steps of: production of a smear preparation; fixation; staining; and drying must be carried out, and thus complicated operations, as well as sufficient knowledge and experience are required. Therefore, training in microscopic observation has been performed for malaria testing as a national project in many endemic countries under instructions of the WHO.
Moreover, the method disclosed in Patent Document 1 necessitates complicated operations, and special apparatus such as a flow cytometer is required.
Accordingly, it cannot be said that any of these test methods could be readily carried out in endemic areas of malaria.
On the other hand, fatty acid binding proteins (FABPs) are known to be a group of proteins having a molecular weight of about 15 kD, are present in cytosol, and are able to bind to a fatty acid. The physiological functions of these proteins are considered to participate in the regulation of metabolic enzyme systems by transfer or accumulation of fatty acids within the cells, but the detailed physiological activities of these proteins have not been clarified yet. There have been known at least seven molecules of FABP such as liver-type (L-FABP), intestine-type (I-FABP), heart muscle-type (H-FABP), brain-type (B-FABP), cutaneous/epidermal-type (C-FABP/E-FABP), fat cell-type (aP2), peripheral neuron-type (myelin-P2), etc., and the primary structures thereof have been determined. All of these FABPs are able to bind to a fatty acid, and are recognized to have a region in which part of the sequence has been duly conserved, so that it is considered that they are members of a family evolved from the common ancestor genes. However, each FABP has a different primary structure as a whole, and shows a unique histologic distribution pattern. The nomenclature of FABP such as liver-type and intestine-type refers to the organ in which such FABP was found first, but does not mean that such an FABP is present exclusively in such organs.
In recent years, it has become known that L-FABP expression in hepatocytes relates to the liver stage development (exoerythrocytic development) of malaria parasites, and that down-regulation of L-FABP expression impairs malaria parasites growth, while overexpression of L-FABP promotes growth (see Non-patent Document 3).
However, in order to apply such findings to methods for malaria testing, L-FABP expression in hepatocytes have to be determined, and thus, they are not practical as test methods. In addition, although Non-patent Document 3 is a report concerning the exoerythrocytic development stage, no symptoms are present in the host during the exoerythrocytic development stage, which may be clinically referred to as a latent period. Therefore, it is also not practical as a test method in this respect. Furthermore, the extent of infection with malaria cannot be known from this test method, in contrast to the methods including microscopic observation of a smear preparation, and the like.
Accordingly, no convenient method for malaria testing which focuses on a L-FABP has been known so far.
Non-patent Document 1: Basic Malaria Microscopy, World Health Organization, 1991
Non-patent Document 2: Yukio Yoshida, “Human Parasitology Illustrated”, Sixth edition, Nanzando Co., Ltd., 2002
Patent Document 1: Japanese Unexamined Patent Publication No. 2006-304774
Non-patent Document 3: Sebastian A. et al., L-FABP is a Critical Host Factor for Successful Malaria Liver Stage Development, Int. J. Parasitol 37: 483-489 (2007)
Non-patent Document 4: Kamijo A. et al., Urinary Liver-type Fatty Acid Binding Protein as a Useful Biomarker in Chronic Kidney Disease, J. Mol. Cell. Biochem. 284: 175-182 (2006)